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Proc Natl Acad Sci U S A. 2016 Jan 5;113(1):E23-31. doi: 10.1073/pnas.1522687112. Epub 2015 Dec 17.

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR.

Author information

1
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139.
2
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139 tabaker@mit.edu.

Abstract

Peroxide operon regulator (PerR) is a broadly conserved hydrogen peroxide sensor in bacteria, and oxidation of PerR at its regulatory metal-binding site is considered irreversible. Here, we tested whether this oxidation specifically targets PerR for proteolysis. We find that oxidizing conditions stimulate PerR degradation in vivo, and LonA is the principal AAA+ (ATPases associated with diverse cellular activities) protease that degrades PerR. Degradation of PerR by LonA is recapitulated in vitro, and biochemical dissection of this degradation reveals that the presence of regulatory metal and PerR-binding DNA dramatically extends the half-life of the protein. We identified a LonA-recognition site critical for oxidation-controlled PerR turnover. Key residues for LonA-interaction are exposed to solvent in PerR lacking metal, but are buried in the metal-bound form. Furthermore, one residue critical for Lon recognition is also essential for specific DNA-binding by PerR, thus explaining how both the metal and DNA ligands prevent PerR degradation. This ligand-controlled allosteric mechanism for protease recognition provides a compelling explanation for how the oxidation-induced conformational change in PerR triggers degradation. Interestingly, the critical residues recognized by LonA and exposed by oxidation do not function as a degron, because they are not sufficient to convert a nonsubstrate protein into a LonA substrate. Rather, these residues are a conformation-discriminator sequence, which must work together with other residues in PerR to evoke efficient degradation. This mechanism provides a useful example of how other proteins with only mild or localized oxidative damage can be targeted for degradation without the need for extensive oxidation-dependent protein denaturation.

KEYWORDS:

Lon; degradation signals; oxidative damage; proteolysis

PMID:
26677871
PMCID:
PMC4711837
DOI:
10.1073/pnas.1522687112
[Indexed for MEDLINE]
Free PMC Article

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